Title: Serum Antibody Repertoire Analysis for High-throughput Epitope Discovery and Characterization

Advisor: Patrick Daugherty

Abstract: 

The serum antibody repertoire is a unique repository of information regarding past immune encounters. Antibodies from the repertoire associated with infection and disease can serve as diagnostic biomarkers upon detection. However, many disease-specific antibodies and their target epitopes remain undiscovered. Therefore, diagnostic reagents must be developed to specifically bind and detect these antibodies. We developed and applied high-throughput methods to discover and characterize peptide epitopes from immune-related diseases. Peptides are suitable reagents as they can often mimic native antibody epitopes with high affinity and specificity. Moreover, the sequences of antibody-binding peptides can be used to identify the original antibody targets, revealing previously unknown antigens that contribute to disease progression and creating new opportunities for therapeutic development.

Here, a large bacterial display peptide library composed of randomized peptides was designed and constructed to screen against human serum specimens and identify epitopes associated with disease. For each specimen, millions of antibody-binding peptide sequences were determined using next-generation sequencing and analyzed computationally to reveal disease-specific binding motifs. This methodology was employed to discover and characterize epitopes from two highly similar viruses, herpes simplex virus type 1 and type 2. We then applied this screening methodology to age-related macular degeneration (AMD) to identify reagents for detecting antibodies associated with an increased risk of advanced AMD. Lastly, a method was developed to selectively deplete highly abundant antibodies from a serum specimen to reduce the complexity of the antibody repertoire and improve the discovery of rare epitopes. Ultimately, these tools are capable of analyzing the antibody repertoire at great depth and can be applied to a broad range of immunological diseases for the discovery of epitopes and the development of novel diagnostics, vaccines, and therapeutics.